Improvement of the 3'-5' exonuclease activity of Taq DNA polymerase by protein engineering in the active site

Mol Cells. 1997 Jun 30;7(3):419-24.

Abstract

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method. Taq DNA polymerase has a domain at its amino terminus (residue 1 to 291) that has a 5'-3' exonuclease activity, a 3'-5' exonuclease domain in the middle (residue 292 to 423), and a domain at its C-terminus that catalyzes polymerase reactions. Taq DNA polymerase is classified into the polI family which is represented by E. coli DNA polymerase I. The three dimensional structural alignment of 3'-5' exonuclease domains from the polI family, DNA polymerases leads us to understand why Taq DNA polymerase does not carry out proof-reading in the polymerase chain reaction. Three sequence motifs, called ExoI, II, and III must be present in order to carry out proof-reading by the 3'-5' exonuclease reaction in DNA polymerization, but Taq DNA polymerase contains none of them. The key catalytic module in the 3'-5' exonuclease is two metal ions chelated by active-site carboxylic amino acids. In order to render the 3'-5' exonuclease activity in Taq DNA polymerase, a catalytic module was constructured in the active site by protein engineering. The mutant Taq DNA polymerase shows twice as much the 3'-5' exonuclease activity as that of wild-type DNA polymerase.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites / genetics
  • DNA Polymerase I / chemistry
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / metabolism*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / chemistry
  • Exodeoxyribonucleases / genetics*
  • Exodeoxyribonucleases / metabolism*
  • Gene Expression
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Protein Engineering
  • Sequence Homology, Amino Acid
  • Taq Polymerase
  • Thermus / enzymology
  • Thermus / genetics

Substances

  • DNA Polymerase I
  • Taq Polymerase
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V